The invention relates to the field of medicine and in particular to vectors for delivery of nucleic acids into cells and to vectors useful for gene therapy.
One of the foremost obstacles to the practical implementation of human gene therapy is the lack of an optimal method for the direct delivery of therapeutic genes to quiescent tissues in vivo. A number of vector systems based on viral components have been developed; however, of these individual virus vector systems, none is optimal and each system displays significant drawbacks.
Retroviruses as vehicles for the delivery of genes into eukaryotic cells have several advantages (Hwang and Gilboa, 1984; Varmus, 1988): 1) gene transfer is relatively efficient; 2) stable integration into the host cell DNA is a natural part of the retroviral life cycle, and therefore the integrated provirus is passed on to all daughter cells, and continues to direct the nonlytic production of its encoded products; and 3) replication-defective vectors can be created by deletion of essential viral genes, which renders the vectors incapable of secondary infection (Mann et al., 1983; Markowitz et al., 1988; Miller and Buttimore, 1986). In spite of these advantages, retroviral gene transfer in its current form has several drawbacks. Most retroviral vectors in current use are traditionally based on Moloney murine leukemia virus (MLV), which requires cell division during infection so that the nucleocapsid complex can gain access to the host cell genome, and hence cannot infect non-dividing cells (Mulligan, 1993; Varmus, 1988). Many cell types are considered to be largely quiescent in vivo, and furthermore, most retroviral vectors are produced from packaging cells at titers on the order of only 106xe2x88x927 colony-forming units (cfu) per ml, which is barely adequate for transduction in vivo. Therefore, retroviral gene transfer in vivo is inefficient, and the traditional approach which has been adopted for retroviral vectors has been to transduce primary cells in culture by the ex vivo method, followed by re-implantation of the transduced cells. This approach requires surgical acquisition, isolation, and culture of autologous cells, and thus is labor-intensive and invasive, and limits the scope of ex vivo retroviral gene transfer to those cell types that can be readily accessed, maintained and manipulated in culture, and reimplanted, e.g., hematopoietic cells, skin fibroblasts, and hepatocytes.
On the other hand, adenoviral vectors have been shown to efficiently infect many cells types in vivo by direct injection. However, as the adenoviral vector remains episomal and does not integrate into the host cell genome, transgene expression is transient. The utility of adenoviral vectors is further limited by cellular and humoral immune responses against wild type adenovirus gene products, which appear to be expressed at low levels in the transduced cells due to xe2x80x9cleakyxe2x80x9d expression despite deletion of the E1 regulatory region (Engelhardt et al., 1993; Yang et al., 1995). Once sensitized, a neutralizing antibody response usually precludes repeat administration by the same vector, and adenovirus-infected cells are soon eliminated by cytotoxic T lymphocytes after transduction (Roessler et al., 1995; Yang et al., 1995). Thus, neither type of virus vector can achieve efficient and long term transduction by direct injection in vivo.
Another virus vector which has been considered is the adeno-associated virus (AAV) (Flotte et al., 1993). AAV was initially thought to be advantageous because it appeared to efficiently infect non-dividing cells (Flotte et al., 1994), and would also undergo site-specific integration into the host cell genome, resulting in long term transduction. However, although these do appear to be attributes of wild type AAV, it seems that these characteristics may not be associated with replication-defective AAV vectors, from which the AAV structural genes, especially the rep gene, have been deleted (Halbert et al., 1995). Other disadvantages of the AAV system have been the limited packaging capacity, only about 4 kilobases, of the vector, and the difficulty of making high titer AAV stocks.
Retrotransposons are mobile genetic elements that insert into new genomic locations by a mechanism that involves reverse transcription of an RNA intermediate. Among the most well-characterized human retrotransposons are L1 elements or LINEs (long interspersed nuclear elements); these non-LTR elements are present in approximately 100,000 copies in the human genome, although 97% of these are functionally inactive due to truncations and rearrangements, and of the remaining 3000 or so full length L1 elements (Singer et al., 1993), it has been estimated that only about 1.5-2.5%, i.e., 30 to 60 copies, are active in retrotransposition (Sassaman et al., 1997). A 6 kb L1 consensus sequence has been derived by sequence analysis of multiple elements (Scott et al., 1987), containing a 5xe2x80x2 untranslated region with an internal promoter (Minakami et al., 1992; Swergold, 1990), two non-overlapping reading frames (ORF 1 and ORF 2), a 3xe2x80x2 untranslated region and 3xe2x80x2 polyadenylated tail; ORF 1 encodes a 40 kD nucleic acid binding protein that co-localizes with L1 mRNA in a cytoplasmic complex (Hohjoh and Singer, 1996; Holmes et al., 1992), while ORF 2 encodes a protein with reverse transcriptase (RT) activity (Hattori et al., 1986; Xiong and Eickbush, 1990) and an N-terminal endonuclease (EN) domain (Feng et al., 1996). Recently, it has been demonstrated that a reporter cassette, with a selectable marker gene driven by the SV40 promoter, can be inserted in reverse orientation into the 3xe2x80x2 untranslated region of L1 elements, and when transfected into cells as an EBNA/oriP-containing episomal plasmid, this system can be used to detect retrotransposition events (Moran et al., 1996; Sassaman et al., 1997). The human L1/reporter element was also active in mouse fibroblasts, suggesting that cellular factors involved in retrotransposition are conserved (Moran et al., 1996). Furthermore, this system was used to characterize novel human L1 sequences that were screened from a genomic library; one of these, L1.3, retrotransposed at a considerably higher frequency, about 1 retrotransposition event scored per 150 cells containing the episomal plasmid (Sassaman et al., 1997). In fact, the actual frequency is probably even higher, as the assay system scored only retrotransposition events occurring in cells that had been pre-selected for the presence of the full length episomal plasmid. Interestingly, it was found that the promoter in the 5xe2x80x2 untranslated region could be replaced with the CMV promoter without significantly affecting the retrotransposition frequency, and that the 3xe2x80x2 untranslated region could be completely deleted without any deleterious effect. When some of the integration sites of the L1/reporter element were cloned and the 5xe2x80x2 junctions sequenced, the elements were found to have been variably truncated 5xe2x80x2 of the selectable marker gene. This results in an integrated element that is presumably incapable of further retrotransposition, as: 1) the 5xe2x80x2 promoter is truncated, thus no mRNA intermediate would be transcribed in the forward orientation; 2) the essential ORF (at least ORF 1, and in some cases ORF 2 also) functions are deleted; and 3) even if the ORF 1 and ORF 2 gene products were to be provided in trans, it has been suggested that the retrotransposition process might be designed to ensure that only mRNA that is in cis with the ORFs is preferentially retrotransposed, perhaps by interaction of the nascent ORF 2 protein with the polyA tail of its own transcript during translation (Boeke, 1997).
Although use of retrotransposons as gene delivery vehicles has been previously suggested (Hodgson et al., 1997; Kingsman et al., 1995), and in fact retrotransposons such as rat VL30 elements have been found capable of being packaged and transmitted by MLV (Chakraborty et al., 1994; Torrent et al., 1994), thus far the efficiency of delivering retrotransposon-encoded sequences to target cells has been the rate-limiting step.
Thus, heretofore there has been no optimal method for direct gene transfer and permanent transduction of quiescent tissues in vivo. Although retroviral gene transfer is currently one of the most commonly used methods for delivery of therapeutic genes, it suffers from problems such as relatively low titers and inability to transduce non-dividing cells; conversely, although adenoviral vectors and non-viral lipid-DNA conjugate vectors offer advantages such as high titers, and the ability to transduce quiescent cells, neither is capable of efficient integration or permanent transduction. Furthermore, other integrating elements such as retrotransoposons and AAV have been modified for use as vectors, but these systems suffer from the lack of an adequate delivery system or simple methods for production of high titer preparations.
A different approach that has been taken in the design of vectors suitable for gene therapy is the combination of elements from distinct viral vectors. Insertion of retroviral structural genes into Herpes simplex virus (HSV) (Savard et al., 1997) has been described. In this case, only retroviral structural genes were inserted into the HSV carrier, which was used to mobilize a retroviral vector sequence already integrated into an indicator cell line.
Insertion of retroviral structural genes and vector constructs into adenovirus (Bilbao et al., 1997) has been reported; however, retroviral structural genes and retroviral vector constructs had to be inserted separately into standard E1-deleted adenovirus vectors (Bilbao et al., 1997), reflecting the limited cloning capacity, about 7 kb, of the adenovirus vectors used. Adenoviruses carrying the retrovirus structural genes and those carrying the retroviral vector constructs were mixed together to achieve co-infection by both types of adenovirus carriers and thus co-expression of retroviral structural gene and vector constructs, resulting in the secondary production of fully assembled, functional retroviral vectors.
Insertion of retroviral structural gene sequences into adenoviral vectors to produce a hybrid construct previously has also been described as a means to achieve efficient transient expression of packaging proteins, particularly for high titer production of vectors pseudotyped with the VSV-G envelope protein, which is toxic to cells and is usually difficult to express in stable packaging cell lines without tight regulation (Yoshida et al., 1997). Other groups have reported similar approaches for efficient production of AAV vectors, by insertion of AAV structural gene or vector sequences into adenovirus-based hybrid expression systems (Fisher et al., 1996; Thrasher et al., 1995). There has been one report describing the production of hybrid vectors consisting of AAV sequences inserted into a Herpes simplex virus (HSV) amplicon for use as a novel gene delivery vehicle (Johnston et al., 1997). Nevertheless, the applicability of retrovirus sequences as inserts within the context of a larger heterologous virus as a vector for gene delivery was heretofore unknown.
Recently, helper-dependent adenoviral vector systems have been developed; the first such system was originally reported by one of us in 1995 (Mitani et al., 1995) and consisted of a reporter gene cassette inserted in an adenoviral genome that had been deleted of many of its structural elements, retaining the inverted terminal repeat (ITR) and packaging signal sequences. Subsequently, a 28 kb vector DNA containing the full length dystrophin gene, with only 360 bp of adenoviral DNA including the replication origin and the packaging signal, was successfully rescued and propagated in adenoviral virions in the presence of helper virus (Clemens et al., 1996; Kochanek et al., 1996). In this system, all the coding sequences that could be toxic or immunogenic to the host were thus removed from the vector DNA. Although some contaminating helper adenovirus is still present in preparations of helper-dependent vectors, cesium chloride gradient separation has allowed purification of the helper-dependent vectors with residual helper virus present at levels of less than 1% (Kochanek et al., 1996; Mitani et al., 1995), and recently reported refinements in the packaging system appear to reduce the level of helper virus contamination even further, to less than 0.01% (Lieber et al., 1996; Parks et al., 1996).
Another advantage of this system is expanded cloning capacity (up to 38 kb) of foreign DNA into the vector. Interestingly, the minimal packaging size requirement was previously defined as 25 kb or so (Mitani et al., 1995); however, it has recently been shown that smaller vector constructs can also be packaged if concatemerization of the vector sequence occurs, resulting in a multimeric size that is within the 27 to 38 kb packageable size range (Parks and Graham, 1997). This expanded capacity is quite advantageous in the case of large genes; as mentioned above, helper-dependent adenoviral vectors recently have been used to deliver the full-length (14 kb) dystrophin gene into skeletal muscle in cell culture and in vivo (Clemens et al., 1996; Haecker et al., 1996). It is noteworthy that the helper-dependent dystrophin adenovectors appear to elicit no inflammatory reaction in vivo. This lack of inflammatory reaction correlated with prolonged expression of the dystrophin transgene, although there have been reports of both shortened and lengthened transgene expression with other deleted adenovector systems (Gao et al., 1996; Kaplan et al., 1997; Lieber et al., 1996). In spite of the success with helper-dependent adenoviral vectors, this approach is still limited by the inherent lack of stable integration.
The present invention incorporates integrating elements such as retrovirus and retrotransposon vectors as inserts within the context of high capacity helper-dependent adenovirus vectors, and thus constitutes a novel type of hybrid vector system that has not previously been described in the literature.
The invention provides hybrid vectors suitable for the delivery of genetic material or nucleic acid molecules into a cell. The hybrid vectors comprise an adenoviral capsid that delivers a helper-dependent nucleic acid molecule encoding an adenoviral region and other inserted heterologous vector elements such as a retroviral region or transposon region.
The adenovirus capsid that encoats the nucleic acid molecule is provided by a helper aderiovirus. The helper adenovirus can be any adenovirus or adenovirus vector, derived from any serotype, that can provide adenovirus early and late proteins necessary for replication and packaging of the helper-dependent nucleic acid molecule, which is itself incapable of being replicated or packaged in eukaryotic cells in the absence of the helper adenovirus.
The adenovirus region of the nucleic acid molecule of the hybrid vector comprises a helper-dependent or xe2x80x9cguttedxe2x80x9d adenoviral vector. Such vectors lack genes necessary for replication and packaging of the adenovirus and are unable to replicable in the absence of the helper adenovirus that supplies the necessary adenoviral structural elements. The adenoviral region therefore can substantially lack nucleic acid sequences encoding adenoviral structural genes. Nucleic acid molecules of the hybrid vectors contain within the adenoviral region a pair of adenoviral inverted terminal repeat sequences as well as a packaging signal from the adenovirus. The elements of the adenoviral region can be those found in any adenovirus, substantially similar sequences, or combinations of such sequences. In one embodiment, adenoviral regions have sequences substantially similar to those found, for example, in adenovirus serotype 2. In another embodiment, adenoviral regions have sequences substantially similar to those found in adenovirus serotype 5.
The hybrid vector system of the invention transduces cells by a two stage mechanism. In the first adenoviral stage, the inserted vector elements, included in the helper-dependent nucleic acid molecule to be delivered, will be carried by the adenoviral capsid, to then be expressed in the target cells and thereby direct the production of the second stage vectors.
In one embodiment, the inserted vector elements of the invention also contain a second stage retroviral region. The retroviral region has sequences that are substantially similar to those of any suitable retrovirus or retroviral vector or vectors including, but not limited to, oncoretrovirus and lentivirus vectors. A preferred retroviral region contains a packaging component, which consists of virus structural gene sequences substantially similar to the gag, pol and env genes from a retrovirus or from different retroviruses. The retroviral region of the hybrid vector is preferably replication incompetent. Such a retroviral region therefore contains a packaging component that lacks the retroviral packaging signal and hence substantially lacks the ability for the sequences in the packaging component itself to be packaged by its encoded retrovirus proteins. The packaging component of the retroviral region also preferably contains one or more promoters that enhance the expression of the retroviral structural genes included in the construct. The promoter(s) can be one or more retroviral long terminal repeats (LTR) flanking the retroviral structural genes. The promoter(s) also can be heterologous viral promoter(s) such as the SV40 promoter or the CMV promoter, or any other promoter that performs this function, including, for example, tissue specific promoters.
The second stage retroviral region of the inserted vector elements encoded within the nucleic acid molecule of the hybrid vector also contains a retroviral gene transfer component that is capable of being packaged by the retroviral structural proteins encoded by the packaging component of the retroviral region. The sequences encoding the retroviral gene transfer component are preferably contained between two flanking retroviral LTR sequences, and also contain a retroviral packaging signal sequence and any nucleic acid sequence of interest. The flanking LTRs can be those of a single retrovirus. In a preferred embodiment the first and second LTRs have different sequences. Such LTR sequences can be substantially similar to those of different retroviruses or those from a single retrovirus that have been altered to possess minimal sequence homology while maintaining functionality. The packaging signal sequence comprises the cis-acting elements that enable the transcribed retroviral gene transfer component to be packaged by the retroviral structural proteins encoded by the packaging component. Nucleic acid sequences of interest can be any nucleic acid molecule for which delivery is desired, including nucleic acids encoding, for example, genes, cDNAs and various RNA species including, for example, ribozymes, antisense sequences and structural RNAs.
In the secondary stage, the newly expressed retroviral vector elements of the hybrid vector will result in a replication-defective retrovirus vector particle, containing the retroviral structural proteins of the packaging component and the packaged RNA transcript of the retroviral gene transfer component, that will stably transduce additional adjacent cells.
The transposon region has a sequence substantially similar to that of any known retrotransposon or DNA transposon, and can also contain heterologous elements within the transposon region. Such transposons permanently integrate into the genome of the initially transduced cells, and the heterologous elements are contained within the transposon regions, and hence will also be integrated during this process. The heterologous elements can also contain promoter, polyadenylation signal, and/or any other sequences necessary for expression of an operably linked sequence of interest also contained within the heterologous element.